Staphylococcus aureus is an important human pathogen, capable of causing life-threatening infections in a variety of host tissues. Osteomyelitis, an invasive and debilitating infection of bone, is one of the most common manifestations of staphylococcal disease. Indeed, osteomyelitis accounts for approximately 2-3 of every 1,000 admissions to pediatric hospitals in the United States, and complicates up to 25% of open fractures. Bone infections are notoriously refractory to treatment due to widespread antimicrobial resistance and pathogen-induced bone remodeling, which limits penetration of antibiotics into the infectious focus. S. aureus is by far the most common cause of musculoskeletal infection, yet the mechanisms by which staphylococci survive within and ultimately destroy bone are poorly understood. The overarching objective of this proposal is to understand how S. aureus regulates its virulence and metabolic programs to survive within bone during osteomyelitis. Bone, like most mammalian tissues, is inherently hypoxic. Moreover, maintenance of skeletal health requires constant bone turnover by resident bone-forming osteoblasts and bone-resorbing osteoclasts. These skeletal cells, in turn, require a specialized metabolism characterized by high rates of glucose uptake, which is expected to limit the carbon sources available to invading pathogens. In order to better understand how S. aureus thrives within this hypoxic and metabolically unique environment, we created a powerful murine model of osteomyelitis capable of precise quantification of both bacterial burdens and bone turnover. By applying transposon sequencing (TnSeq) to this osteomyelitis model, we identified >200 staphylococcal genes important for survival in bone. Importantly, bacterial responses to hypoxia were found to be critical determinants of survival during osteomyelitis, as hypoxic growth not only dictates the energy production strategies used by staphylococci, but also augments the production of quorum-dependent virulence factors that participate in bone destruction. Based on these preliminary data, we hypothesize that S. aureus survival in bone is facilitated by (a) quorum-regulated virulence factor expression in response to hypoxia, and (b) specific nutrient utilization programs that enable growth in the unique metabolic environment of bone. The proposed Aims will test this hypothesis to determine (i) the mechanism by which hypoxic growth triggers increased staphylococcal virulence, (ii) the metabolic pathways that support bacterial growth in bone, and (iii) the role of host hypoxic signaling pathways in antibacterial immunity and bone remodeling during osteomyelitis. Completion of these studies will elucidate microbial survival strategies during invasive infection, determine the impact of hypoxia on bacterial pathogenesis, and help to meet a critical need for new osteomyelitis therapeutics by defining putative antimicrobial and anti-virulence targets.